The alpha helix is a fundamental structural unit in the fabric of proteins, with 30% of all amino acids in proteins occurring in alpha helices.1 When helical sequences of amino, acids are exposed on an exterior surface of a protein, the helix frequently interacts with another protein, a segment of DNA or of RNA.2,3 This biomolecular recognition is central to a large range of biological processes, for example those summarized in Table 1. In most cases however only a few alpha helical turns are actually involved in the molecular recognition. For example, transcriptional regulators (e.g. p53, NF-kBp65, VP16c)4,6 apoptosis regulators (e.g. Bak)7 and RNA-transporter proteins (e.g. Rev)8 all contain a short alpha helical sequence of only 2-4 turns that mediates function by direct interaction with a receptor.
TABLE 1Some Biological Processes Mediated by Interaction ofAlpha-Helices with Other Biomoleculesα-Helical PeptideBiological targetProcess MediatedReferenceProtein-DNA interactionsZif268G/C rich majorDNA transcription9grooveProtein-RNA interactionsHIV ReverseRev ResponseRNA reverse transcription10TranscriptaseElement (RRE)λ-N peptideBoxB RNATranscriptional anti-10terminationP22 peptidesBoxB RNATranscriptional anti-10terminationProtein-Protein interactionsp53HDM2Tumor Suppressor silencing4BakBcl-XLApoptosis Regulation7VHL peptideElongin CDNA transcription11VP16 activation domainHTAFII31DNA transcription12hPTHhPTHrPCalcium homeostasis13Dynorphin Aκ,δ-OpioidPain signal transmission14, 15receptorsApolipoprotein-ELDL receptorLipid metabolism,16cholesterol homeostasisNeuropeptide-YNPY receptorsMultiple functions17GalaninGal receptorsMultiple functions18CorticotropinCRF receptorsStress responses19Releasing FactorCalcitonin GeneCGRP receptorsMultiple functions20Related PeptideNociceptinORL1 receptorPain transmissionVasointestinalVPAC1 & 2Multiple functions21PeptideNuclear CoactivatorsNuclear ReceptorsDNA Transcription22, 23(eg. SRC1, GRIP1)
Short peptide sequences of less than 15 amino acid residues that correspond to these helical protein regions are not thermodynamically stable structures in water when removed from their protein environments.24,25 Short synthetic peptides corresponding to such alpha helical recognition motifs tend not to display appreciable helical structure in water, away from the helix-stabilizing hydrophobic environments of proteins. If short peptide alpha helices could be stabilized or mimicked by small molecules, such compounds might be valuable chemical or biological probes and lead to development of novel pharmaceuticals, vaccines, diagnostics, biopolymers, and industrial agents. The goal of structurally mimicking short alpha helices with small molecules that have biological activity comparable to proteins has not yet been realized.
Attempts to stabilize short alpha helical peptides have met with limited success to date. Examples of methods used to stabilize alpha helicity in peptides longer than 15 residues are helix-nucleating templates26-29, metals30-35, unnatural amino acids36,37, non-covalent side chain constraints38,39 and covalent side chain linkers (e.g. disulfide-40,41, hydrazone-42, lactam-43-50, aliphatic linkers51-53). Although mimics of short alpha helical segments have remained elusive, some recent attempts have been reported using non-peptidic oligoamide and terphenyl scaffolds that project 2-3 substituents into similar three dimensional space as the side chains of an alpha helix54-56.
Helix nucleating templates are organic molecules at the N- or C-terminus of a peptide which can make hydrogen bonds with the first or last four NH or C═O groups in the peptide, and thus nucleate helicity throughout the rest of the peptide. Such a task is not trivial due to the specific position, pitch and orientation of the required NH or C═O groups. Several attempts have had some success, these include Kemp's triacid, cyclic proline molecules,26, 57-61, Mueller's Cage compound62, Bartlett's cap28, and Kahn's cap63. There have also been some attempts to synthesize capping groups by replacing a hydrogen bond with a covalent link as in the case of Satterthwait's cap64.
Transition metals30-35 are often found in proteins serving both catalytic and structural roles. By exploiting the ability of transition metals such as Cu2+, Zn2+, Cd2+, Ru3, Pd2+ to bind both acidic and basic residues it has been possible to achieve helix stabilization. Chelation of metals to donor groups generally yields ˜1 kcal/mol−1 in helix stabilization, however stabilization is very dependent on solvent, salt concentration and pH.
Unnatural amino acids have also been reported to favor helix stabilization. In general n-alkyl substitution, α,α- and γγ-disubstitution increases helix stability. β,β-Disubstitution reduces helicity, and β-tertiary substitution totally abolishes helix propensity, thus it appears the helix is quite sensitive to steric effects at the beta position65. α-Aminoisobutyric acid (Aib) in particular is known to stabilize α- and 310-helical conformations and has been used to improve the biological activity of several peptides. Nociceptin analogues containing 1 or 2 Aib residues resulted in 10-15 fold increases in potency and affinity (Ki=0.02 nM)66. Similarly an analogue of p53 containing Aib and 1-aminocyclopropanecarboxylic acid (Ac3c) yielded a peptide 1735 more active than the native peptide67. Finally when Aib was substituted into deltorphin-C analogues a 10-fold Ki increase in selectivity was obtained for 8 vs opioid receptor subtypes68 
Disulfide bridges have been employed to stabilize helices via two methods. The first involves the use of a modified, unnatural amino acid D,L 2-amino-6-mercaptohexanoic acid placed at the ith (D) and i+7th (L) residues to stabilize two turns of an alpha helix41. The second approach involves using a D-cysteine (i) and L-cysteine (i+3) disulfide to stabilize a single alpha helical turn. This approach was successful to a certain extent, however the conformation was quite solvent dependent40. It has recently been reported that this approach was used to constrain the SRC-1 peptide, which is known to adopt an alpha helical conformation in the estrogen receptor-α, and inhibit this receptor with a Ki of 25 nM69.
Lactam bridges have often been used to increase helicity and turn conformations in long peptides. They generally involve the covalent amide linkage of the side chains of lysine/ornithine residues with the side chains of aspartic/glutamic acid residues at either i to i+3 or i to i+4 positions. These constraints although initially examined in model peptides have been applied to numerous biological targets in which the bioactive conformation is deemed to be helical. In general this constraint has been employed in relatively long sequences (15-30 residues) generally to create monocyclic analogues, but in some cases, up to three lactam bridges have been included. Some examples of their use include PTH, NPY, CRF, GCN4, Galanin and Dynorphin-A. Despite their inception over 10 years ago, there is still a lack of consensus over which residue combinations are the best, although it appears i to i+4 spacing is optimal for alpha helicity. Early pioneering work by Taylor48 suggested Lys→Asp was the optimal combination, however, later work by Houston identified Glu→Lys as optimal, although this study totally neglected to use aspartic acid70. More recent work by Taylor has involved using overlapping lactam bridges to yield a highly rigid hexapeptide alpha helix, highly resistant to chemical and thermal degradation45, and with some templating capability71. However, this hexapeptide scaffold is limited for general application as a template since only two of six residues are available for interaction with a biological target. The synthesis and properties of side-chain lactam bridged peptides, their alpha helical nature, functional activity and potential for improved proteolysis resistance has recently been reviewed43.
Modified lactam-type bridges can also be spaced i to i+7, therefore requiring longer linkers, and in this regard, aspartic/glutaniic acid, and/or diaminopropionic acid residues provide a convenient functionality to which linkers can be attached. Some of these have included diaminopentane linkers joined to two glutamic acids53, 4-(aminomethyl)-phenylacetic acid linked via aspartic acid and 1,3-diaminopropionic acid49, or alternately 4-(aminomethyl)-phenylazobenzoic acid joined to the N- and C-terminus of an octapeptide. The two former methods resulted in reasonably stable helices, whilst the latter resulted in a 310 helical/random coil conformation depending on the cis/trans isomerization of the azo linkage.
Ring closing metathesis has been used in helix stabilization. Pioneered by Grubbs72, this approach has been utilized with allyl-modified serine/homoserine residues in i→i+4 fashion. It has not been overly successful in stabilizing alpha helicity, although some 310 stabilization was observed. Other approaches have incorporated both S- and R-α-methyl-α-allylglycine, along with the α-homoallyl and α-homohomoallyl derivatives, positioned at either i→i+4 or i→i+751. It was found that the R-isomer at the i position and the S-isomer at the i+7 position, with an 11 carbon link provided 44% helix stability compared to the uncyclized peptide.
Non-peptidic mimicry of alpha helices has been rare, with only a few examples reported. The first reported non-peptidic helix mimetics were 1,1,6-trisubstituted indanes, that when coupled to an amino acid were capable of presenting three side chains in a helical like conformation. When applied as tachykinin mimetics, they had micromolar affinity for NK and NK3 receptors73. These type of molecules were recently applied to magainin mimicry, and whilst they were capable of killing bacterial strains they still maintained high hemolytic activity74. Recently Kahne and co-workers developed a pentasaccharide helix mimetic based on GCN4 which bound DNA with micromolar affinity75. By far the most successful approach to nori-peptidic alpha helix mimicry has been achieved by Hamilton and co-workers who have successfully developed two generic types of molecules—terphenyls and oligoamides capable of mimicking the i, i+4, i+7 side chains on one face of an alpha helix. These mimetics have been successfully applied to inhibition of HIV gp41 mediated viral fusion with an IC50 of 15.7 μg/mL76, and also inhibit Bak/Bcl-XL complex with low micromolar to nanomolar efficiency77,78.
There have been no previous reports of cyclic pentapeptides adopting alpha helices on their own. Usually cyclic pentapeptides have been used to mimic the smaller beta or gamma turns of peptides and proteins. There are numerous examples of cyclic peptides that mimic beta or gamma turns reported in the literature as demonstrated by several reviews73-81. A prime example is synthetic compound 1 which is a cyclic pentapeptide containing the RGD tripeptide sequence. This compound is a potent glycoprotein IIb/IIIa antagonist and orally bioavailable antithrombotic and antitumor agent73, 82, 83. Compound 1 provides a demonstration of how the simple insertion into a cyclopeptide of a rigid amino acid as a copformational constraint can result in favorable biological and pharmacological properties; and a number of its derivatives are in advanced clinical trials. For example, in phase MI clinical trials, the cyclic RGD-containing heptapeptide drug eptifibatide (Integrilin) has been shown to reduce the incidence of cardiac events in patients at risk of abrupt vessel closure after coionary angioplasty84.

Constraints do not need to be complex, as shown in compound 2 where an ornithine (or lysine) side chain is used to form the macrocycle. This constraint, in conjunction with proline and D-cyclohexylalanine constraints, induces intramolecular hydrogen bonding that confers potent antagonism (IC50 10 nM) against human C5a receptors on polymorphonuclear leukocytes both in vitro and in vivo85. C5a antagonists are expected to be useful for combating inflammatory diseases.
Cyclotheonamide A (Compound 3) is a 19-membered cyclic pentapeptide possessing α-keto amide and trans-4-aminobutenoyl constraints. It was isolated from the marine spongeTheonella sp. and was shown to inhibit the serine proteases thrombin (Ki 180 nM) and trypsin (Ki 23 nM). The NMR solution structure of compound 3 was recently found to be the same in water as those found in the solid state when bound to trypsin and thrombin86, suggesting that this natural product is pre-organized for enzyme binding, and that selectivity is associated with the positioning of the D-Phe side chain.
Lactam bridges (i→i+3, i→i+4, i→i+7) have previously been reported to increase alpha helicity in longer peptides, although the literature is very inconsistent about their capacity to do so43-51. There have been no reports of cyclic pentapeptides adopting alpha helical structures.
The synthesis and conformation of multicyclic alpha helical peptides comprising three repeats of a heptapeptide constrained by a side-chain to side-chain lactam bridge in (i)→(i+4) positions has been reported48,114. These studies showed that spaced cyclic moieties in a peptide can induce or stabilize alpha helicity.
Conformational restrictions in the form of (i)→(i+4) lactam bridges incorporated into known peptide sequences to induce helical conformation have also been reported115. Three constrained helical 31-residue peptides derived from human parathyroid hormone and containing 1, 2 or 3 cyclic moieties were shown to be potent agonists of the parathyroid hormone and parathyroid hormone-related protein receptor
There are few studies that report alpha helicity for the theoretical minimum (pentapeptide) sequence needed to define a beta turn, the existence and properties of which are not well defined despite the likelihood that only one or a few turns of a protein helix need to be mimicked for agonist/antagonist biological activity.
There are many commercially important peptides that are known to adopt alpha helical structures that would benefit from improved structural stabilization and improved resistance to proteolysis. Some examples include calcitonin which has been launched for the treatment of osteoporosis, the parathyroid hormone which is in phase II clinical trials for the treatment of osteoporosis, a substance-P/saporin conjugate which is in preclinical trials for the treatment of pain and conantokin-G which is under development for the treatment of epilepsy (Pharmaprojects, 2004).
Accordingly, there is a need for stabilized short peptide alpha helices that can mimic biological molecules or that can be incorporated into non-peptidic or semi-peptidic compounds to mimic biological molecules. Such peptides could potentially be valuable as chemical and biological probes, pharmaceuticals, biotechnology products such as vaccines, or diagnostic agents, new components of biopolymers and industrial agents.